Human serum albumin (HSA) is the most abundant protein of human plasma and performs wide range of functions such as maintaining the plasma oncotic pressure, functioning as an antioxidant, carrying out functions as a universal transport and depot protein with extraordinary ligand binding capacity and modulating fluid distribution; it also displays various important enzymatic and anti-inflammatory activities (Boldt J, British Journal of Anaesthesia 2010; 104(3):276-84). HSA is a valuable biomarker of several diseases. Moreover, clinically HSA has been employed to treat several diseases including hypovolemia, shock, burns, surgical blood loss, trauma, hemorrhage, etc. It is also utilized as an excipient for vaccines, supplement in cell culture medium, carrier of oxygen and in various others biotechnological applications (Kobayashi, Biologicals 34 (2006); 55-59).
Its wide applications and immense therapeutic potential, makes HSA a protein that is highly in demand, and it is estimated that the annual world need for this protein exceeds 500 tons. Currently, HSA is obtained primarily from the fractionation of collected human blood that is a limited and an unsafe source as there exists the risk of it being contaminated by various blood derived pathogens like HIV, hepatitis and the like.
To eliminate the potential risk of contamination, there is an indispensable need to develop non-animal-derived alternatives and low cost methods to obtain large quantities of pathogen free recombinant HSA as a primary substitute for the plasma-derived form (Chen et. al, Biochimica et Biophysica Acta 1830 (2013): 5515-5525).
HSA is a 66.5 kDa monomeric, single chain, non-glycosylated, heart shaped protein made up of three homologous domains containing 17 disulfide bonds and one free sulfhydryl group (Kobayashi, Biologicals 34 (2006): 55-59). The non-glycosylated and single chain polypeptide features of HSA make it less complex than the other blood extracted proteins like plasminogen activator and clotting factors, and has encouraged various research groups to carry out number of attempts for the production of HSA. Over the past couple of decades extensive research is going in the direction, but till yet no host system has been proven to be ideal enough when, rHSA production is the concerned issue, because of their respective limitations. Surprisingly, one of the branches had remained very less explored i.e. rHSA production using E. coli as a host system.
E. coli is one of the most convenient host systems that has contributed in the production of more than 30% approved recombinant pharmaceuticals by FDA (major examples include human insulin, plasminogen activator, growth hormone etc) (Ceccarelli et. al, Frontiers in microbiology (2014); 5 (172)). E. coli derived products have more economical potential as fermentation processes are more economical compared to other expression hosts as it grows rapidly and reach high cell densities using inexpensive and simple substrates (Tripathi et al., Defence Science Journal (2009); 59 (2): 137-146). However, it has not been exploited for rHSA production.
Till date E. coli has not been exploited for the production of recombinant HSA for therapeutic usage. This is due to the fact that a majority of the recombinantly expressed HSA proteins in E. coli host system form aggregates (more than 90% of the expressed rHSA) leading to the formation of inclusion body as previously reported (Lawn, Nucleic Acids Res. 1981; 9:6103-6114; Latta, BioTechnol. 1987; 5: 1309-1314). An attempt to express rHSA in the periplamsic space of the E. coli host system has also been carried out recently (Ghafari et. al, New cellular biotechnology journal (2013); 3 (9)) but it explores only a preliminary narrow era with the prime goal to check the status of expression of rHSA in a periplasmic space and reveals no more in addition to that.
A recent process developed by the inventors results in the recovery of 60% of the total expressed rHSA in the soluble fraction. (Patent application filed no. 201611027096, entitled—“Process for production, and isolation of recombinant human serum albumin in E. coli”). The process was based on the modulation of the cellular growth parameters followed by osmolytic assistance at a crucial cell lysis step of rHSA preparation from E. coli host system. However, a limitation of the method is that some fraction of proteins obtained from the soluble fraction is not functionally active and display lower levels of activity as compared to ideal situations.